Tissue engineering - bone grafting developments

On first inspection, use of synthetic bone graft substitutes appears to be Polyfiller for bones. However, it is one of the most advanced and practical forms of tissue engineering available.

Bone grafting is the practice of taking a sample of a patient’s bone (usually from the illiac crest in the pelvis), bone from a human bone bank (which has been de-cellularised, screened for transmittable diseases and sterilised), or a synthetic substitute (either in bulk, granule, putty or cement form), and implanting it into a bone injury to promote healing. It is applied in situations where healing is not expected to occur spontaneously or might be attenuated because of the injury’s severity. These are known technically as critical defect or non-union sites.

The role of the graft is to stimulate the recruitment of mesenchymal stem cells that can differentiate into osteoblasts (specialised bone forming cells). As they infiltrate the graft, they differentiate, proliferate and start to lay down healthy bone tissue. Once the graft site becomes living tissue, it can begin to remodel with bone resorbing cells (osteoclasts) working in tandem with a second wave of osteoblasts. Ideally, the graft site’s structure eventually becomes seamlessly blended with the surrounding bone. Repair and healing is identical to that seen with ‘green stick’ fractures, where a fracture is splinted in a plaster cast and allowed to heal. In time, the load bearing ability of the bone is restored fully.

Compatible combination

Replacement of damaged bone tissue is not a modern concept, both ancient Egyptian and Chinese records show the use of foreign materials to augment or replace it. The technique did not prove reliable until recently, most commonly gangrene and blood poisoning proved to be fatal complications. The modern clinical success rate owes much to the development of aseptic techniques.

Today, the field of synthetic bone graft development is interdisciplinary, encompassing the expertise of materials scientists, medical engineers, bone biologists, pathologists and orthopaedic surgeons. This has produced a wide variety of candidate materials, ranging from tantalum metal foams to collagen sponges. Most are used to provide a porous material that can act as a scaffold for cellular colonisation, however, some are designed to work alone, harnessing the body’s regenerative capacity through chemical or topographical interactions with the graft material. Others work with biological agents, such as stem cells or growth factors, to stimulate healing and ultimately restore structure and function.

Development of bone graft substitute materials began in the late 1960s, but owed much to earlier observations. The success of the Charnley Hip replacement resulted in an awareness of the need to match physical materials properties with the right engineering solution.

Initially biocompatibility was concerned with the degree to which a material did not stimulate a bad response from the host, that is its inertness. While an ‘active’ response to implanted materials was associated with a toxic or allergenic response, poisoning or inflaming the surrounding host tissues. In extreme cases, the responses could become systemic, having fatal consequences. The concept that bioactive materials could be developed that might result in a positive specific interaction between product and host is a relatively recent trend.

The right chemistry

Containing up to 70wt% bone mineral, bone is a complex calcium-phosphate rich compound with a similar crystal structure to hydroxyapatite (Ca10 (PO4)6(OH)2). It also contains varying levels of trace element minerals, such as sodium, zinc, magnesium, strontium, potassium, silicon and a significant carbonate content.

Crystallographic characterisation of the mineral or ceramic component of bone in 1964 led to significant interest in developing calcium-phosphate ceramic-based graft materials, in particular hydroxyapatite. However, their poor strength initially limited their usefulness to coatings and composite fillers for improved biocompatibility. It was work in the late 1960s on bioactive glasses (a group of calcium-rich silicate-based glasses) that popularised the concept of bioactivity. Yet, in applications such as those concerned with blood handling, stimulation of a response (coagulation and clot formation) is undesirable.

The concept of biocompatibility, therefore, had to be application specific – considering the response of the host to a material that was appropriate for that application, in addition to the appropriate response of the material to the host environment.

In a suture material, for example, under some circumstances, a controlled rate of resorption is desirable to save the patient having their stitches removed. Similarly, the bone cells, with time to affect a seamless repair, should remodel a bone graft substitute material. In both cases, it is clear that a too rapid or slow rate of resorption would be undesirable, and the degradation products, as well as the bulk material, must be biocompatible.

While various bone graft materials are used, in many cases, understanding of the mechanisms by which these materials support or stimulate bone regeneration is limited. This reflects the fact that biomaterials development was hindered by the limited choice of analytical tools to assess host and materials responses. Graft materials were developed through incisive thought and gut feelings. New analytical tools, combined with developments in the basic materials, and biological and surface sciences, are expected to rapidly progress understanding.

The surface physiochemistry of the more successful bioactive synthetic bone graft materials appears to be hydrophilic in nature and promotes the adsorption of various bone formation friendly (osteogenic) proteins, which facilitate cell adhesion, proliferation and maturation. There is also some evidence that the development of a calcium-phosphate layer, known as ‘bone-like apatite,’ is also critical to the bioactivity of some materials, and that the release of bioavailable ions from a materials surface can directly stimulate cell metabolism and function. Control of a synthetic bone graft’s resorbability and bioactivity, via control of the surface chemistry, is therefore key to clinical success (see image adjacent).

Several different approaches have been used to enhance bioactivity, such as combining hydroxyapatite with the more resorbable tricalcium phosphate to facilitate faster remodelling, or substituting trace element ions such as magnesium, strontium and silicon into bioactive glasses, glass ceramics, various calcium phosphates and hydroxyapatite. The latter fine tunes surface physiochemistry and facilitates the release of beneficial ion species.

Further system optimisation will require more precise knowledge about the way in which blood and serum proteins interact competitively with a material’s surface under physiological conditions, along with information about the temporal sequence in which key proteins are critical to stimulating bone healing cascade. This is in conjunction with the processes of ion exchange occurring at the interface and with circulating cells.

Specific structure

The importance of macroporosity (pores with a nominal diameter of between 20-2,000µm) was illustrated by Sam Hulbert and co-workers in the early 1970s. By implanting a series of macroporous ceramic and polymeric materials, they demonstrated that fibrous encapsulation could be reduced as porosity increases, and that the pore interconnection size and distribution are critical to the penetration and maintenance of healthy bone into the scaffold.

There are two properties that explain this behaviour – the influence of porosity on the local biomechanics and structural permeability. Bone cells are sensitive to load, enabling them to adapt to the environment for maximum efficiency. A dense scaffold structure will effectively screen any bone tissue of load (known as stress shielding and a common complication with metallic hip implants), reducing the normal stimulus for the tissue to develop or be maintained in situ. Bone, being a dense mineralised tissue, is highly dependant on the presence of an interpenetrating vascular network to maintain its oxygen potential and nutrient supply. Pores and pore interconnections must therefore be of sufficient size and occur with the right frequency to enable penetration (see main image, top).

The presence of an interconnected microporosity or strut porosity also has a profound effect on the bioactivity of bone grafts, resulting in the formation of a greater volume of new tissue more rapidly when implanted in bony sites. However, muscle (in the absence of mature bone-forming cells) grafts with strut porosity are able to induce bone formation (by their influence on local mesenchymal stem cells in the muscle tissue). Although there will certainly be some biomechanical and permeable influence as a result of the strut porosity, which may encourage greater and more rapid bone formation, this does not explain the inductive phenomenon. There is evidence that the introduction of porosity at this scale has an effect through its influence on surface physiochemistry and fluid mechanics, altering both protein and ion dynamics at the graft surface.

Additionally, the increased roughness and inclusion of surface concavities at the micrometre scale, associated with the presence of strut porosity, introduces cell-scaled surface features that may enhance cell attachment and mobility at the interface (see image adjacent).

The sensitivity of cells to the topography of their environment is well documented and has demonstrated topographical cell guidance and sensitivity of cells to the scale of topographical features using silicon wafer micropatterning techniques to remove chemical variation as a confounding variable. Introducing more strut porosity may affect local cell populations by providing them with an environment both biochemically and morphologically that triggers a bone-forming response.

To progress, researchers will need to fully understand the interactions that occur between inorganic ceramic scaffolds and the local physiological environment that are capable of guiding stem cell differentiation and bone regeneration.